Current diagnostic measures for Chronic Kidney Disease (CKD) include detection of reduced estimated glomerular filtration rate (eGFR) and albuminuria, which have suboptimal accuracies in predicting disease progression. The disease complexity and heterogeneity underscore the need for multiplex quantification of different markers. The goal of this study was to determine the association of six previously reported CKD-associated plasma proteins [B2M (Beta-2-microglobulin), SERPINF1 (Pigment epithelium-derived factor), AMBP (Protein AMBP), LYZ (Lysozyme C), HBB (Hemoglobin subunit beta) and IGHA1 (Immunoglobulin heavy constant alpha 1)], as measured in a multiplex format, with kidney function, and outcome. Antibody-free, multiple reaction monitoring mass spectrometry (MRM) assays were developed, characterized for their analytical performance, and used for the analysis of 72 plasma samples from a patient cohort with longitudinal follow-up. The MRM significantly correlated (Rho = 0.5–0.9) with results from respective ELISA. Five proteins [AMBP, B2M, LYZ, HBB and SERPINF1] were significantly associated with eGFR, with the three former also associated with unfavorable outcome. The combination of these markers provided stronger associations with outcome (p < 0.0001) compared to individual markers. Collectively, our study describes a multiplex assay for absolute quantification and verification analysis of previously described putative CKD prognostic markers, laying the groundwork for further use in prospective validation studies.

Equal volume (2 μL) of plasma samples containing approximately 100 μg of total protein were used for LC-MRM-MS analysis. Briefly, after protein denaturation (8 M urea), reduction (10 mM dithioerythritol) and alkylation (50 mM iodoacetamide) the samples were digested with trypsin [(1:100 enzyme: protein ratio (w/w)] for 16 hours in the dark (RT). The peptide mixture was desalted with solid phase extraction zip-tips (Thermo Scientific) and the extracted peptides were dried using a vacuum centrifuge. The dried peptides were solubilized in mobile phase A (97.9% H2O, 2% acetonitrile, 0.1% formic acid), pH 3.5 to obtain a final concentration of 0.5 μg/μL. A mixture of the SIS peptides was then added in each sample after drying the peptides as follows: IGHA1: 8000 ng/mL, B2M: 800 ng/mL, HBB: 770 ng/mL, AMBP: 2240 ng/mL, LYZ: 800 ng/mL, SERPINF1: 668 ng/mL. Liquid chromatography was performed using an Eksigent nano-HPLC system, coupled with a C18 analytical column (75 μm × 150 mm, particle size 5 μm, pore size 100 Å) (Thermo Scientific). Peptide separation and elution were performed with a 60 min gradient of 5–90% mobile phase B (80% acetonitrile v/v, 0.1% FA, 19.9% H2O) at a flow rate of 300 nL/min. Samples were injected into the LC system and loaded on the C18 column. Tryptic peptides were analyzed on an AB Sciex 4000QTRAP with a nanoelectrospray ionization source controlled by Analyst 1.5 software (AB Sciex). The mass spectrometer was operated in MRM mode, with the first (Q1) and third quadrupole (Q3) at 0.7 unit mass resolution. Data analysis was performed using the Skyline software. Savitzky-Golay smoothing was applied. The top signal producing transition was selected as the quantifier transition in all cases, while the remaining transitions were used as qualifier transitions, for accurate peak profile and retention time confirmation.

Human plasma samples from chronic kidney disease (CKD) patients (CKD stages: 2-3-4-5).